Transforming growth factor- (TGF-) is involved in many aspects of cancer biology, including proliferation, apoptosis, migration, and metastasis. TGF-'s effects are mediated through a family of cell surface receptors, including the type III TGF- receptor (TRIII), which can mediate downstream signaling. TRIII is lost in many human cancers, including non-small cell lung cancer (NSCLC), with loss correlated with more aggressive disease and disease progression. TRIII also undergoes ectodomain cleavage, producing a soluble protein (sTRIII) that may sequester ligand away from the cell surface receptors and prevent activation of TGF- signaling. We have demonstrated that the loss of TRIII shedding, generated through a single base pair mutation in TRIII (M740A), increases TGF- signaling, and inhibits tumorigenicity and metastasis in vivo. However, the mechanism of TRIII shedding and the relative contributions of sTRIII versus cell-surface TRIII to cancer initiation and progression have not been investigated and remain a fundamental barrier to developing novel therapeutic agents that target sTRIII/TRIII and TGF- superfamily signaling pathways. Protease predictions from PROSPER and preliminary data using TAPI-2, a broad spectrum inhibitor of members of the matrix metalloproteinase (MMP) and a disintegrin and metalloprotease (ADAM) families, suggest that TRIII shedding is mediated by proteases in the MMP or ADAM family. This project will seek to identify the proteases involved in TRIII shedding using a combination of loss (shRNA, inhibitors) and gain of function (expression, recombinant protein) approaches. We will also investigate the biological contributions of soluble and cell- surface TRIII to anchorage-independent cell growth, migration, invasion, proliferation, and TGF- signaling in lung adenocarcinoma cell lines that are deficient in TRIII cleavage. To study the in vivo contributions of sTRIII to cancer initiation and progression, we are developing an in vivo TRIIIM70A/M740A;FSF-KrasG12D/+ lung carcinoma model in which there is a loss of sTRIII production. We anticipate that loss of TRIII shedding will delay tumorigenesis while promoting tumor growth and metastasis after tumor initiation due to the dual effects of TGF- as a tumor suppressor early in carcinogenesis and then later, as a tumor promoter. This work will define the roles of cell surface and sTRIII in carcinogenesis by providing a mechanistic understanding of the protease(s) involved in the generation of sTRIII and the contributions of sTRIII to lung adenocarcinoma development and progression, which may lead to the development of new therapeutic strategies that target TRIII shedding or TGF superfamily signaling pathways.